The influence of single and multiple applications of pyrene on the evolution of pyrene catabolism in soil

The influence of pyrene added in a single application (0, 50, 100 and 200 mg kg(-1)) was investigated in multiple applications (1 x 50, 2 x 50 and 4 x 50 mg kg(-1)) on the evolution of catabolic activity in a pristine pasture soil. The microbial community's ability to degrade pyrene was assessed at 0, 4, 8 and 12 weeks by the mineralization of added 14C-pyrene. Significant mineralization (>5%) of added 14C-pyrene only occurred after 4 weeks soil-pyrene contact time in most of the pyrene-amended soils. Pyrene-amended soils showed statistically significantly shorter (P<0.05) lag times compared to the control soil after 8 and 12 weeks soil-pyrene contact time. Further, the rates of degradation increased in the presence of pyrene, peaking at 8 weeks. In terms of the overall extents of pyrene mineralization, there were statistically significant increases (P<0.05) between 4 and 8 weeks, with little difference between 8 and 12 weeks, with the general trend that an increase in pyrene concentration resulted in higher levels of mineralization. Increasing the concentration and number of pyrene additions can have a significant impact on the adaptation of the soil microflora to degrade pyrene over time.     

Limited microbial degradation of pyrene metabolites from the estuarine polychaete Nereis diversicolor

We compared microbial mineralization of [4,5,9,10-14C]pyrene and its eukaryotic [4,5,9,10-14C]pyrene metabolites in estuarine sediments. Metabolites were obtained by exposing the estuarine deposit-feeding polychaete Nereis diversicolor to sediment-associated 14C-pyrene, followed by homogenization of the worms and extraction of the pyrene-metabolites. In sediment from a pristine Danish Fjord only 2.6% of the added metabolite-label and 1.7% of the pyrene-label were mineralized to 14CO2 during 175 days incubation. Pre-exposure of the pristine sediment to unlabelled pyrene for 60 days increased the mineralization potential for 14C-pyrene substantially, as 81.2% was mineralized to 14CO2 during 95 days incubation, whereas 14C-pyrene metabolite label was unaffected by pre-exposure to pyrene. In comparison, naturally aged bunker-oil contaminated sediment did not show elevated potentials for mineralization of neither 14C-pyrene nor 14C-metabolites. Six bacterial strains of known pyrene degraders were tested for growth on crystalline 1-hydroxypyrene. 1-Hydroxypyrene is the only intermediate eucaryotic metabolite of pyrene. The results indicate that 1-hydroxypyrene was not utilized as a sole source of carbon and energy by any of them. In addition, respiration was depressed in all six strains when exposed to crystalline 1-Hydroxypyrene, demonstrating an acute toxic effect of 1-hydroxypyrene. The results presented here suggest that microbial degradation of pyrene is not enhanced by release of aqueous and polar metabolites by marine invertebrates.     

Biodegradation of PAHs in soil: Influence of chemical structure, concentration and multiple amendment

The influence of PAH chemical structure and concentration, added in either single (75 or 300 mg kg⁻¹) or multiple (2 × 75, 2 × 150 or 4 × 75 mg kg⁻¹) applications as single- or multiple-contaminant systems, on the development of PAH biodegradation in a pristine soil was investigated. Development in microbial catabolic ability was assessed at 0, 28, 56 and 84 d by monitoring ¹⁴C-naphthalene, ¹⁴C-phenanthrene and ¹⁴C-pyrene mineralisation over 14 d in respirometric assays. The presence of other contaminants influenced the ability of the indigenous microflora to mineralise structurally different contaminants over time. ¹⁴C-Naphthalene mineralisation was inhibited by the presence of other contaminants; whereas the presence of naphthalene significantly enhanced rates of mineralisation in multiple-contaminant systems containing ¹⁴C-phenanthrene and ¹⁴C-pyrene. Generally, increasing the number of contaminant applications has implications for catabolic activity of soil microbes. It is suggested the toxic nature of PAHs retarded mineralisation at increased contaminant concentrations.     

Fate of pyrene in contaminated soil amended with alternate electron acceptors

Creosote-contaminated soil samples from the Libby Ground Water Contamination Superfund Site in Libby, MT, were amended with the potential alternate electron acceptors (AEA) nitrate (KNO3), manganese oxide (MnO2), and amorphous iron oxyhydroxide (FeOOH) and incubated at low oxygen tensions (0-6% O2). The fate of 14C-pyrene was evaluated with respect to the different soil amendments. The fate of 14C from the radiolabeled pyrene with regard to mineralization and bound residue formation within soil humic fractions was not significantly different from controls for the iron and manganese amended soils. Nitrate amendments appeared to stimulate 14C-pyrene mineralization at a level of 170 mg NO3-N kg(-1), and inhibit mineralization at 340 mg NO3-N kg(-1). The stimulatory effect did not appear to be the result of nitrate serving as an electron acceptor. Although AEA amendments did not significantly affect the rate or extent of 14C-pyrene mineralization, results of oxygen-deprived incubations (purged with N2) indicate that AEA may be utilized by the microbial community in the unsaturated contaminated soil system.     

Comparison of microbial pyrene and benzo[a]pyrene mineralization in liquid medium, soil slurry, and soil

The microbial degradation of 14C-pyrene and 14C-benzo[a]pyrene by a bacterial mixed culture was studied within a mixture of the PAHs phenanthrene, anthracene, pyrene, fluoranthene, and benzo[a]pyrene as sole carbon source in the different culture systems: (i) liquid medium, (ii) soil slurry (surface and grinding influence), and (iii) soil. The fate of these two labeled compounds was followed in these systems with an emphasis on mineralization to carbon dioxide, extractability, and adsorption to humic materials and formation of unextractable residual. Mineralization showed the most obvious differences: soil slurries achieved the best results both concerning the extent of mineralization and the time required. The highest extent of pyrene mineralization (54% within 21 days) was observed in soil slurries; in liquid media, pyrene mineralization was slower, but reached approximately the same extent (54% in 150 days); in soils, mineralization reached only 36% of added pyrene after 160 days. Benzo[a]pyrene was mineralized in a mixture of PAHs in soil slurries to an extent of 34% within 70 days, whereas mineralization in liquid medium and soil occurred in the range of 5% (70 days). Mineralization of benzo[a]pyrene in sand slurries was lower compared to soil slurries (19% in sand slurries vs. 32% in soil slurries within 50 days).     

Comparison of Microbial Pyrene and Benzo[a]Pyrene Mineralization in Liquid Medium,Soil Slurry,and Soil

The microbial degradation of ¹⁴C-pyrene and ¹⁴C-benzo[a]pyrene by a bacterial mixed culture was studied within a mixture of the PAHs phenanthrene, anthracene, pyrene, fluoranthene, and benzo[a]pyrene as sole carbon source in the different culture systems: (i) liquid medium, (ii) soil slurry (surface and grinding influence), and (iii) soil. The fate of these two labeled compounds was followed in these systems with an emphasis on mineralization to carbon dioxide, extractability, and adsorption to humic materials and formation of unextractable residual. Mineralization showed the most obvious differences: soil slurries achieved the best results both concerning the extent of mineralization and the time required. The highest extent of pyrene mineralization (54% within 21 days) was observed in soil slurries; in liquid media, pyrene mineralization was slower, but reached approximately the same extent (54% in 150 days); in soils, mineralization reached only 36% of added pyrene after 160 days. Benzo[a]pyrene was mineralized in a mixture of PAHs in soil slurries to an extent of 34% within 70 days, whereas mineralization in liquid medium and soil occurred in the range of 5% (70 days). Mineralization of benzo[a]pyrene in sand slurries was lower compared to soil slurries (19% in sand slurries vs. 32% in soil slurries within 50 days).     

Mineralisation of target hydrocarbons in three contaminated soils from former refinery facilities

This study investigated the microbial degradation of ¹⁴C-labelled hexadecane, octacosane, phenanthrene and pyrene and considered how degradation might be optimised in three genuinely hydrocarbon-contaminated soils from former petroleum refinery sites. Hydrocarbon mineralisation by the indigenous microbial community was monitored over 23 d. Hydrocarbon mineralisation enhancement by nutrient amendment (biostimulation), hydrocarbon degrader addition (bioaugmentation) and combined nutrient and degrader amendment, was also explored. The ability of indigenous soil microflora to mineralise ¹⁴C-target hydrocarbons was appreciable; ≥16% mineralised in all soils. Generally, addition of nutrients or degraders increased the rates and extents of mineralisation of ¹⁴C-hydrocarbons. However, the addition of nutrients and degraders in combination had a negative effect upon ¹⁴C-octacosane mineralisation and resulted in lower extents of mineralisation in the three soils. In general, the rates and extents of mineralisation will be dependent upon treatment type, nature of the contamination and adaptation of the ingenious microbial community.     

Microbial bioavailability of pyrene in three laboratory-contaminated soils under aerobic and anaerobic conditions

Changes in bioavailability of pyrene in three uncontaminated soils were examined under aerobic and anaerobic conditions. Three soils were aerobically aged with pyrene and [(14)C]pyrene for 63 days, then incubated with water, nitrate, or sulfate under aerobic or anaerobic conditions for one year. Under aerobic conditions, microorganisms in two soils mineralized 58-82% of the added [(14)C]pyrene. The two soils amended with nitrate were seen to have enhanced aerobic mineralization rates. In one of these soils, non-extractable pyrene was seen to decrease over the course of the study due to desorption and mineralization, nitrate amendment enhanced this effect. Under anaerobic conditions, generated with a N(2):CO(2)(g) headspace, two soils with nitrate or sulfate amendment showed an increase in extractable [(14)C]pyrene at 365 days relative to inhibited controls, presumably due to microbially mediated oxidation-reduction potential and pH alteration of the soil environment. These observations in different soils incubated under aerobic and anaerobic conditions have important implications relative to the impact of microbial electron acceptors on bioavailability and transport of non-polar organic compounds in the environment suggesting that, given enough time, under the appropriate environmental conditions, non-extractable material becomes bioavailable. This information should be considered when assessing site specific exposure risks at PAH contaminated locations.     

Impact of carbon nanomaterials on the behaviour of 14C-phenanthrene and 14C-benzo-[a] pyrene in soil

The impact of fullerene soot (FS), single-walled (SWCNTs) and multi-walled (MWCNTs) carbon nanotubes on the behaviour of two ¹⁴C-PAHs in sterile soil was investigated. Different concentrations of carbon nanomaterials (0, 0.05, 0.1 and 0.5%) were added to soil, and ¹⁴C-phenanthrene and ¹⁴C-benzo[a]pyrene extractability assessed over 80 d through dichloromethane (DCM) and hydroxypropyl-β-cyclodextrin (HPCD) shake extractions. Total ¹⁴C-PAH activity in soils was determined by combustion, and mineralisation of ¹⁴C-phenanthrene was monitored over 14 d, using a catabolically active pseudomonad inoculum. No significant loss of ¹⁴C-PAH-associated activity from CNM-amended soils was observed over the ‘aging’ period. CNMs had a significant impact on HPCD-extractability of ¹⁴C-PAHS; extractability decreased with increasing CNM concentration. Additionally, ¹⁴C-phenanthrene mineralisation was inhibited by the presence of CNMs at concentrations of ≥0.05%. Differences in overall extents of ¹⁴C-mineralisation were also apparent between CNM types. It is suggested the addition of CNMs to soil can reduce PAH extractability and bioaccessibility, with PAH sorption to CNMs influenced by CNM type and concentration.     

Influence of root-exudates concentration on pyrene degradation and soil microbial characteristics in pyrene contaminated soil

The effect of ryegrass (Lolium perenne L.) root-exudates concentration on pyrene degradation and the microbial ecological characteristics in the pyrene contaminated soil was investigated by simulating a gradually reducing concentration of root exudates with the distance away from root surface in the rhizosphere. Results showed that, after the root-exudates were added 15 d, the pyrene residue in contaminated soil responded nonlinearly in the soils with the same pyrene contaminated level as the added root-exudates concentration increased, which decreased first and increased latter with the increase of the added root-exudates concentration. The lowest pyrene concentration appeared when the root exudates concentration of 32.75 mg kg(-1) total organic carbon (TOC) was added. At the same time, changes of microbial biomass carbon (MBC, C(mic)) and microbial quotient (C(mic)/C(org)) were opposite to the trend of pyrene degradation as the added root-exudates concentration increased. Phospholipid fatty acid (PLFA) analysis revealed that bacteria was the dominating microbial community in pyrene contaminated soil, and the changing trends of pyrene degradation and bacteria number were the same. The changing trend of endoenzyme-dehydrogenase activity was in accordance with that of soil microbe, indicating which could reflect the quantitative characteristic of detoxification to pyrene by soil microbe. The changes in the soils microbial community and corresponding microbial biochemistry characteristics were the ecological mechanism influencing pyrene degradation with increasing concentration of the added root-exudates in the pyrene contaminated soil.     

The impact of synthetic pyrethroid and organophosphate sheep dip formulations on microbial activity in soil

Sheep dip formulations containing organophosphates (OPs) or synthetic pyrethroids (SPs) have been widely used in UK, and their spreading onto land has been identified as the most practical disposal method. In this study, the impact of two sheep dip formulations on the microbial activity of a soil was investigated over a 35-d incubation. Microbial utilisation of [1-(14)C] glucose, uptake of (14)C-activity into the microbial biomass and microbial numbers (CFUs g(-1) soil) were investigated. In control soils and soils amended with 0.01% sheep dip, after 7d a larger proportion of added glucose was allocated to microbial biomass rather than respired to CO(2). No clear temporal trends were found in soils amended with 0.1% and 1% sheep dips. Both sheep dip formulations at 0.1% and 1% concentrations resulted in a significant increase in CFUs g(-1) soil and [1-(14)C] glucose mineralisation rates, as well as a decline in microbial uptake of [1-(14)C] glucose, compared to control and 0.01% SP- or OP-amended soils. This study suggests that the growth, activity, physiological status and/or structure of soil microbial community may be affected by sheep dips.     

The extractability and mineralisation of cypermethrin aged in four UK soils

Cypermethrin is a widely used insecticide that has caused concern due to its toxicity in the aquatic environment. As with all land applied pesticides, the most significant source of water pollution is from the soil, either due to leaching or washoff. The behaviour of cypermethrin in the soil controls the likelihood of future pollution incidents, with two of the most significant processes being the formation of bound residues and microbial degradation. The formation of bound residues and mineralisation was measured in four organically managed soils from the UK. The formation of bound residues was measured using three different extraction solutions, 0.01 M CaCl₂, 0.05 M HPCD and acetonitrile. Biodegradation was assessed by measurement of mineralisation of cypermethrin to CO₂. The formation of bound residues varied according to extraction method, soil type and length of ageing. In two of the four soils studied, acetonitrile extractability decreased from 100% initially to 12-14% following 100 d ageing. The extent of mineralisation increased after 10-21 d ageing, reaching 33% of remaining activity in one soil, however following 100 d ageing the extent of mineralisation was significantly reduced in three out of the four soils. As with the formation of bound residues, mineralisation was impacted by soil type and length of ageing.     

Biomineralisation of 1,2,4-trichlorobenzene in soils by an adapted microbial population

In laboratory experiments the mineralisation of 14C-labelled 1,2,4-trichlorobenzene (1,2,4-TCB) in soils was studied by direct measurement of the evolved 14CO2. The degradation capacity of the indigenous microbial population was investigated in an agricultural soil and in a soil from a contaminated site. Very low mineralisation of 1% within 23 days was measured in the agricultural soil. Whereas in the soil from the contaminated site the mineralisation occurred very fast and in high rates; up to 62% of the initially applied amount of 1,2,4-TCB were mineralised within 23 days. The transfer of the adapted microbial population into the agricultural soil significantly enhanced the mineralisation of 1,2,4-TCB in this soil, reflecting, that the transferred microbial population survived and maintained its degradation ability in the new microbial ecosystem. Additional nutrition sources ((NH4)2HPO4) increased the mineralisation rates in the first days significantly in the contaminated soil. In the soil from the contaminated site high amounts of non extractable 14C-residues were formed.     

The formation of bound residues of diazinon in four UK soils: implications for risk assessment

The behaviour of diazinon in the soil determines the likelihood of further pollution incidents, particularly leaching to water. The most significant processes in the control of the fate of diazinon in the soil are microbial degradation and the formation of bound residues. Soils from four sites in the UK were amended with diazinon and its ¹⁴C labelled analogue and incubated for 100 days. After 0, 10, 21, 50 and 100 days, the formation of bound residues was assessed by solvent extraction, and the microbial degradation of diazinon by mineralisation assay. In microbially active soils, diazinon is degraded rapidly, reducing the risk of future pollution incidents. However, where there was limited mineralisation there was also significantly lower formation of bound residues, which may lead to water pollution via leaching. The formation of bound residues was dependent on extraction type. Acetonitrile extraction identified bound residues in all soils, with the bound residue fraction increasing with increasing incubation time.     

Atrazine is not readily mineralised in 24 temperate soils regardless of pre-exposure to triazine herbicides

Mineralisation of atrazine in soil has been shown to depend on previous exposure of the herbicide. In this study, 24 Danish soils were collected and screened for potential to mineralise atrazine. Six soils were chosen, because they had never been exposed to atrazine, whereas 18 soils were chosen because of their history of application of atrazine or the related compound terbuthylazine. None of the 24 soils revealed a mineralisation potential of more than 4% of the added atrazine within a 60 day timeframe. In an atrazine adapted French soil, we found 60% mineralisation of atrazine in 30 days. Cattle manure was applied in order to boost the microbial activity, and a 2-3% increase in the atrazine mineralisation was found in some of the temperate soils, while in the highly adapted French soil it caused a 5% reduction.     

The influence of a NAPL on the loss and biodegradation of 14C-phenanthrene residues in two dissimilar soils

This study was carried out to assess the influence of diesel, applied over a log concentration range, on the loss and extractability of phenanthrene (measured as putative 14C-phenanthrene residues) in two different soils. The influence of diesel on the ability of a cyclodextrin based extraction method to predict the microbial bioavailability of 14C-residues was also assessed. An increase in loss of 14C-residues with increasing diesel concentration from 0 to 2000 mg kg-1 was generally observed with time in both soils. It is suggested that this trend is attributable to competitive sorption for soil sorption sites and to a lesser extent to displacement of 14C-residues from soil sorption sites by diesel resulting in greater compound availability and therefore greater loss by degradation via the actions of indigenous microorganisms. However, in the 20000 mg kg-1 diesel treatments of both soils, results indicated a delayed loss. It is suggested that this retarded loss was due to the formation of a discrete NAPL-phase into which 14C-phenanthrene residues partitioned, thereby decreasing their availability and as a consequence their degradation. Furthermore, it is suggested that nutrient limitation may have slowed down degradation rates as diesel concentrations increased. Comparison between cyclodextrin-extractability and microbial mineralisation supported the use of cyclodextrin to assess microbial bioavailability of 14C-residues after 50 d or more ageing up to diesel concentrations of 2000 mg kg-1. However, results suggested that at high diesel concentrations (specifically 20000 mg kg-1) co-extraction of 14C-phenanthrene residues may have occurred as a result of the combined solvation powers of both the cyclodextrin and the diesel. Furthermore, mineralisation of 14C-phenanthrene residues may have been affected by extreme nutrient limitation in this treatment.     

Pyrene degradation by yeasts and filamentous fungi

The saprotrophic soil fungi Fusarium solani (Mart.) Sacc., Cylindrocarpon didymum (Hartig) Wollenw, Penicillium variabile Sopp. and the yeasts Rhodotorula glutinis (Fresenius) Harrison and Rhodotorula minuta (Saito) Harrison were cultured in mineral medium with pyrene. The remaining pyrene concentrations were periodically determined during 20 incubation days, using HPLC. To assess the metabolism of pyrene degradation we added 0.1 microCi of [4,5,9,10] 14C-pyrene to each fungi culture and measured the radioactivity in the volatile organic substances, extractable, aqueous phase, biomass and 14CO2 fractions. The assays demonstrated that F. solani and R. glutinis metabolized pyrene as a sole source of carbon. Differences in their activities at the beginning of the cultures disappeared by the end of the experiment, when 32 and 37% of the original pyrene concentration was detected, for the soil fungi and yeasts, respectively. Among the filamentous fungi, F. solani was highly active and oxidized pyrene; moreover, small but significant degradation rates were observed in C. didymum and P. variahile cultures. An increase in the 14CO2 evolution was observed at the 17th day with cosubstrate. R. glutinis and R. minuta cultures showed similar ability to biotransform pyrene, and that 35% of the initial concentration was consumed at the end of the assay. The same results were obtained in the experiments with or without glucose as cosubstrate.     

Chemical assays of availability to earthworms of polycyclic aromatic hydrocarbons in soil

The bioavailable concentration of an organic pollutant is less than the concentration determined by vigorous extraction of soil. Because bioavailability varies with the particular compound, soil, and aging time, an assay for bioavailability is needed. Three methods were tested: extraction with a 25% aqueous solution of tetrahydrofuran (THF), 95% ethanol, and C18 membranes. Evaluations were conducted with a mixture of four polycyclic aromatic hydrocarbons (PAHs) added to five dissimilar soils and with pyrene as sole PAH added to six soils, and the availability of aged and freshly added compounds was determined. Assimilation by earthworms (Eisenia fetida) was used to assess bioavailability. For extraction with THF and earthworm uptake, the correlation coefficients of determination (R2) for anthracene, chrysene, pyrene, and benzo(a)pyrene added as a mixture exceeded 0.85. The R2 values for assays with the C18 membrane were 0.77 or higher. The values for pyrene added alone were 0.710 and 0.823, respectively. R2 values for assays with ethanol often exceeded 0.87, but lower values were sometimes obtained. We suggest that such solvent or solid-phase extractions may be useful in assessing PAH bioavailability.     

Fractionation of heavy metals and distribution of organic carbon in two contaminated soils amended with humic acids

The effects of humic acids (HAs) extracted from two different organic materials on the distribution of heavy metals and on organic-C mineralisation in two contaminated soils were studied in incubation experiments. Humic acids isolated from a mature compost (HAC) and a commercial Spaghnum peat (HAP) were added to an acid soil (pH 3.4; 966 mg kg(-1) Zn and 9,229 mg kg(-1) Pb as main contaminants) and to a calcareous soil (pH 7.7; 2,602 mg kg(-1) Zn and 1,572 mg kg(-1) Pb as main contaminants) at a rate of 1.1g organic-C added per 100g soil. The mineralisation of organic-C was determined by the CO(2) released during the experiment. After 2, 8 and 28 weeks of incubation the heavy metals of the soils were fractionated by a sequential extraction procedure. After 28 weeks of incubation, the mineralisation of the organic-C added was rather low in the soils studied (<8% of TOC in the acid soil; <10% of TOC in the calcareous soil). Both humic acids caused significant Zn and Pb immobilisation (increased proportion of the residual fraction, extractable only with aqua regia) in the acid soil, while Cu and Fe were slightly mobilised (increased concentrations extractable with 0.1M CaCl(2) and/or 0.5M NaOH). In the calcareous soil there were lesser effects, and at the end of the experiment only the fraction mainly related to carbonates (EDTA-extractable) was significantly increased for Zn and decreased for Fe in the humic acids treated samples. However, HA-metal interactions provoked the flocculation of these substances, as suggested by the association of the humic acids with the sand fraction of the soil. These results indicate that humic acid-rich materials can be useful amendments for soil remediation involving stabilisation, although a concomitant slight mobilisation of Zn, Pb and Cu can be provoked in acid soils.     

Intrinsic and induced isoproturon catabolic activity in dissimilar soils and soils under dissimilar land use

The catabolic activity with respect to the systemic herbicide isoproturon was determined in soil samples by (14)C-radiorespirometry. The first experiment assessed levels of intrinsic catabolic activity in soil samples that represented three dissimilar soil series under arable cultivation. Results showed average extents of isoproturon mineralisation (after 240 h assay time) in the three soil series to be low. A second experiment assessed the impact of addition of isoproturon (0.05 microg kg(-1)) into these soils on the levels of catabolic activity following 28 days of incubation. Increased catabolic activity was observed in all three soils. A third experiment assessed levels of intrinsic catabolic activity in soil samples representing a single soil series managed under either conventional agricultural practice (including the use of isoproturon) or organic farming practice (with no use of isoproturon). Results showed higher (and more consistent) levels of isoproturon mineralisation in the soil samples collected from conventional land use. The final experiment assessed the impact of isoproturon addition on the levels of inducible catabolic activity in these soils. The results showed no significant difference in the case of the conventional farm soil samples while the induction of catabolic activity in the organic farm soil samples was significant.